Delivery of packaged rna to mammalian cells

ABSTRACT

Described herein are compositions relating to alphavirus-based virus-like particles (VLPs) and methods for making and using the described VLPs. The described compositions include VLPs and vectors and cells used to produce the VLPs. Also included are related methods to produce the VLPs, to transduce cells using the VLPs, and to produce a protein or polynucleotide of interest in a target cell using the VLPs. Also described are alphavirus-based replicons that allow for expression of proteins or polynucleotides of interest in a target cell without a cytopathic effect.

CROSS REFERENCE TO RELATED APPLICATIONS

The application is a divisional of U.S. patent application Ser. No.15/190,992, filed Jun. 23, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/388,441, filed Sep. 26, 2014, now U.S. Pat. No.9,506,041, which claims the benefit of application numberPCT/US2013/031876, filed Mar. 15, 2013, which claims the benefit of U.S.Provisional Application No. 61/615,687 filed Mar. 26, 2012, which ishereby incorporated by reference in its entirety.

TECHNICAL FIELD

The invention described herein relates to delivering and transcribingrecombinant polynucleotides to mammalian cells usingreplication-defective virus-like particles.

BACKGROUND

Alphaviruses belong to the group IV Togaviridae family of viruses. Thealphaviruses are small, spherical, enveloped viruses with a genome of asingle positive sense strand RNA. The total genome length ranges between11,000 and 12,000 nucleotides, and has a 5′ cap, and 3′ poly-A tail. Thefour non-structural protein genes (NSP genes) are encoded in the 5′two-thirds of the genome, while the three structural proteins aretranslated from a subgenomic mRNA colinear with the 3′ one-third of thegenome.

There are two open reading frames (ORFs) in the alphavirus genome,non-structural and structural. The first includes NSP genes and encodesproteins (nsP1-nsP4) necessary for transcription and replication ofviral RNA. The second encodes three structural proteins: the corenucleocapsid protein C, and the envelope proteins P62 and E1 thatassociate as a heterodimer. The viral membrane-anchored surfaceglycoproteins are responsible for receptor recognition and entry intotarget cells through membrane fusion.

The Sindbis (and VEEV) virus is an alphavirus whose genome comprises apositive mRNA strand of 11703 nucleotides. This virus infects a varietyof vertebrate hosts. The genome of Sindbis virus encodes nonstructural(NS, replicon) and structural proteins (capsid and pH dependentfusogenic envelope) that are directly translated in the cytoplasm of thehost cell. The alphaviruses also include Aura virus, Babanki virus,Barmah Forest virus, Bebaru virus, Cabassou virus, Chikungunya virus,Eastern equine encephalitis virus, Everglades virus, Fort Morgan virus,Getah virus, Highlands J virus, Kyzylagach virus, Mayaro virus, Me Trivirus, Middelburg virus, Mosso das Pedras virus, Mucambo virus, Ndumuvirus, O'nyong-nyong virus, Pixuna virus, Rio Negro virus, Ross Rivervirus, Salmon pancreas disease virus, Semliki Forest virus, Southernelephant seal virus, Tonate virus, Trocara virus, Una virus, Venezuelanequine encephalitis virus, Western equine encephalitis virus, andWhataroa virus.

Infection of host cell with an alphavirus results in cytotoxicityculminating with apoptosis, This is mostly due to both: expression ofalphavirus genomic RNA in large quantities triggering antiviral state inhost cells and direct interaction of alphaviral non-structural proteins( NSP2 of SIN or NC of VEEV) with cellular mRNA synthesis ortranslational shut-off causing cytophathic effect (CPE) on host cellhost cell. A natural Sindbis virus variant containing a point mutationin one of the nonstructural proteins, NSP2 (at position 726)demonstrated sustained and noncytopathic growth in infected cellsalthough the viral titer recovered from infected cells was substantiallyreduced (Frolov, I. et al., J. Virol. 3845-65 (May, 1999)).

Alphaviruses are of interest to gene therapy researchers. Ross Rivervirus, Sindbis virus, Semliki Forest virus (SFV), and Venezuelan equineencephalitis virus (VEEV) have all been used to develop vectors for genedelivery. Pseudotyped viruses may be formed by combining alphaviralenvelopes glycoproteins and retroviral capsids. Alphaviral envelopeglycoproteins pseudotyped retroviruses or lentiviruses are able tointegrate the genes that they carry into the potential host cells. Thepseudotyped alphaviruses are recognized and infected by the alphaviralenvelope proteins E2 and E1. Stable integration of viral genes ismediated by retroviral interiors of these vectors.

There are limitations to the use of alphaviruses in the field of genetherapy due to their lack of specificity of targeting. However, throughthe introduction of variable antibody domains in a non-conserved loop inthe structure of E2, specific populations of cells have been targeted.Furthermore, the use of whole alphaviruses for gene therapy is oflimited efficacy both because several internal alphaviral proteins areinvolved in the induction of apoptosis upon infection and also becausethe alphaviral capsid mediates only the transient introduction of mRNAinto host cells. Neither of these limitations extends to alphaviralenvelope pseudotypes of retroviruses or lentiviruses.

SUMMARY

One aspect of the description is a virus-like particle (VLP) comprisingan alphavirus replicon, wherein the alphavirus replicon comprises arecombinant polynucleotide, a retroviral gag protein, a fusogenicenvelope protein, in which the VLP does not contain an alphavirusstructural protein gene. The alphavirus replicon may be derived fromSindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, and NSP4,and a retroviral packaging signal. The retroviral gag protein may bederived from Rous sarcoma virus or murine leukemia virus. The fusogenicenvelope protein is selected from the group consisting ofhaemagglutinin, Rous sarcoma virus (RSV) fusion protein, an E protein oftick borne encephalitis virus and dengue fever virus, the E1 protein ofSFV, baculovirus gp64, and Vesicular stomatitis (Indiana) virus-G(VSV-G) protein, preferably a glycoprotein, or fragment or derivativethereof, more preferably from a RNA virus or a retrovirus, or fragmentor derivative thereof, most preferably VSV-G or EnvA, or an alterationof VSV-G. The VLP described herein may be capable of binding to aeukaryotic cell, preferably a human cell. The binding of the VLP may bespecific to a target cell. The VLP described herein preferablyreplicates in the target cell. In some embodiments the VLP describedherein is not cytopathic to the cell. The recombinant polynucleotide ofthe VLP may comprise a miRNA, shRNA or an antisense RNA, preferably ashRNA or antisense RNA that knocks down expression of a gene in thecell. The recombinant polynucleotide of the VLP may comprise an RNAencoding a protein that can be expressed by the cell.

Another embodiment of the description is a method of producing the VLPdescribed herein, comprising the steps of co-transforming a eukaryoticcell with a first vector comprising a polynucleotide sequence encodingthe alphavirus replicon, wherein the alphavirus replicon includes thepolynucleotide of interest, a second vector comprising a polynucleotidesequence encoding the retroviral gag protein, and a third vectorcomprising a polynucleotide sequence encoding the fusogenic envelopeprotein; culturing the co-transformed cell under conditions suitable tocause each vector to produce its encoded product, thereby producing theVLP, and isolating the VLP from the cell, wherein neither the vectorsnor the cell contain any alphavirus structural protein genes.

Another embodiment of the description is kit comprising a first vectorcomprising a polynucleotide sequence encoding an alphavirus replicon,wherein the alphavirus replicon includes the polynucleotide of interest;a second vector comprising a polynucleotide sequence encoding theretroviral gag protein; and a third vector comprising a polynucleotidesequence encoding the fusogenic envelope protein, such as VSV-G. In someembodiments, the vectors provided with the kits do not includealphavirus structural protein genes. Alternatively, in some embodimentsone or more of the alphavirus replicon, retroviral gag protein andfusogenic envelope protein may be encoded by the same vector.

Another embodiment of the description is a method of expressing therecombinant polynucleotide in a eukaryotic cell comprising treating acell with the VLP described herein.

Another embodiment of the description is a method of delivering therecombinant polynucleotide described herein to a subject, comprisingadministering to said subject the VLP described herein.

Another embodiment of the description is a method of treating orpreventing a disease or a disorder in a subject, comprisingadministering to a subject the VLP described herein. Preferably,expression of the gene of interest supplements deficient expression ofan endogenous gene by said subject.

Another embodiment of the description is a pharmaceutical compositioncomprising the VLP described herein.

Another embodiment of the description is a eukaryotic cell produced bytreating the cell with a VLP described herein.

To produce VLPS of this sort several components may be produced bytransfecting or nucleofecting one or more vectors encoding thesecomponents into a cell line for in vitro production. In someembodiments, these components are encoded by separate vectors to reducethe likelihood that the resulting VLP will be replication competent. Forexample, a multi-vector system may be used where one vector encodes thegenetic material, such as an alphavirus-based RNA polynucleotide, to bepackaged by the VLP; another encodes the structural proteins of the VLP,such a gag protein; and another vector encodes a fusion protein, such asVSV-G, to facilitate fusion of the VLP to the membrane of a target cell.The alphavirus-based RNA polynucleotide can be derived from anyalphavirus. In some embodiments, the RNA polynucleotide is derived fromSindbis virus and encodes Sindbis virus nonstructural protein. In someembodiments, the RNA polynucleotide is derived from Venezuelan equineencephalitis virus (VEEV) and encodes VEEV nonstructural proteins.However, other alphavirus nonstructural proteins may suffice for the RNAconstruct described herein. Suitable alphaviruses include Aura virus,Babanki virus, Barmah Forest virus, Bebaru virus, Cabassou virus,Chikungunya virus, Eastern equine encephalitis virus, Everglades virus,Fort Morgan virus, Getah virus, Highlands J virus, Kyzylagach virus,Mayaro virus, Me Tri virus, Middelburg virus, Mosso das Pedras virus,Mucambo virus, Ndumu virus, O'nyong-nyong virus, Pixuna virus, Rio Negrovirus, Ross River virus, Salmon pancreas disease virus, Semliki Forestvirus, Southern elephant seal virus, Tonate virus, Trocara virus, Unavirus, Venezuelan equine encephalitis virus, Western equine encephalitisvirus, and Whataroa virus.

Also described herein are cells comprising the vectors described toproduce VLPs. These cells may be used to produce the VLPs describedherein by transcribing or expressing the polynucleotides of the vectors.For instance, a mammalian cell transfected with a vector having apolynucleotide sequence encoding an alphavirus RNA construct having agene or polynucleotide of interest and a packaging signal, a vectorencoding a retroviral gag protein, and a vector encoding a viral fusionprotein could produce a VLP having the expressed viral fusion protein onits surface with one or two copies of the encoded alphavirus RNAconstruct housed inside the VLP. Furthermore, because none of thesevectors encode alphavirus structural proteins the possibility ofcreating an infectious virus is substantially reduced compared tosystems that do include alphavirus structural proteins.

VLPs produced using the vectors and cells are also described herein. TheVLPs described herein will have four general characteristics: they willcomprise one or two RNA molecules encoding an alphavirus replicon, andoptionally a protein of interest; they will have a viral core comprisinga retroviral gag protein, or, in some embodiments, a gag fusion protein;they will have a surface protein to facilitate fusion with a cell, andthey will not contain a polynucleotide that encodes an alphavirusstructural protein.

The VLPs described herein may be produced in a variety of ways, as willbe apparent to those skilled in the art based on the provideddisclosure. The commonality to these various methods is the expressionof the described vectors in a cell capable of expressing the necessaryproteins (gag and a fusion protein) and producing the alphavirus-basedRNA replicon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C illustrate the elements of the three vectorsexemplified herein. FIG. 1A shows the plasmid pCMV-Sin Rep-protein ofinterest-2 (POI-2), which is a Sindbis virus-based replicon. FIG. 1Bshows the plasmid pGAG-protein of interest-1 (POI-1), which encodes aretroviral gag protein. FIG. 1C shows the plasmid pEnvelope, whichencodes the viral fusion protein, such as VSV-G.

FIG. 2 schematically shows an example of a vector according to thedescription corresponding to the plasmid depicted in FIG. 1A. The DNAsequence of the vector is provided in the Sequence Listing, appendedhereto.

FIG. 3 schematically represents how the transducing VLPs describedherein are produced.

FIGS. 4A, 4B and 4C depict the results of three separate VLPtransduction experiments. FIG. 4A shows cells transduced with aGFP-expressing Sindbis virus replicon packaged into VLPs obtained fromsupernatants collected from Baby Hamster Kidney (BHK)-21 cellstransfected with all three plasmids; FIG. 4B shows the absence of GFPexpression in cells incubated with cell supernatants collected fromBHK-21 cells nucleofected with only the pCMV-Sin Rep-POI-2 andpGAG-POI-1 plasmids in the absence of envelope protein. FIG. 4C showstransduction of human embryonic kidney (HEK293T) cells to demonstratethat the constructed VLPs can transduce human cells.

FIGS. 5A, 5B, 5C and 5D show that the VLPs described herein cansuccessfully transduce cells following storage at 4° C. for 1 hour (FIG.5A), 2 days (FIG. 5B), 4 days (FIG. 5C), and 8 days (FIG. 5D).

FIGS. 6A and 6B show the expression levels of Gaussia luciferase bycells transduced with VLPs having a VEEV replicon capable of expressingthe Gaussia luciferase gene (FIG. 6A, condition 4). Conditions 1, 2, and3 of FIG. 6A show expression of Gaussia luciferase by cells transducedwith VEEV replicons having no exogenous gene (condition 1) or with agene encoding GFP (conditions 2 and 3). FIG. 6B provides an illustrationof the expression kinetics of the luciferase protein during the first 4hours after transduction.

FIGS. 7A and 7B illustrate the genetic process by the cre/lox system canbe used to alter gene expression in a cell by delivering cre recombinaseto a cell via transduction with a VLP having a VEEV replicon with thecre recombinase gene (FIG. 7A). The gene expression profile for a cellline engineered to express GFP in the absence of cre recombinase and RFPin the presence of cre recombinase is shown before and after (days 5, 6,and 7) transduction with a VLP carrying a replicon capable of expressingcre recombinase. FIG. 7B shows results of delivering functional crerecombinase (red cells) to cells engineered to express GFP in theabsence of cre recombinase.

FIG. 8 depicts expression of GFP following transduction of BHK-21 cells,293T cells, or NIH3T3 cells with VLPs having either a Sindbis virus orVEEV-based replicon capable of expressing GFP. 24 and 48-hour timepoints are shown for all samples.

FIGS. 9A, 9B, 9C, 9D, 9E, 9F and 9G illustrate functional packaging ofnon-coding RNA into VLPs. Cells transduced with VLPs containing:VEE-rep-GFP (encoding GFP) (FIG. 9A), VEE-Rep miGFP (encoding microRNAfor GFP) (FIG. 9B), VEERep-GFP and VEERep miGFP (simultaneoustransduction) (FIG. 9C), VEERep-GFP and VEERep miGFP (miRNA transductionoccurred 4 hours prior to GFP transduction) (FIG. 9D), VEE-rep-Cre (usedin place of scrambled miRNA to demonstrate specificity of miRNA) (FIG.9E), VEE-Rep-GFP and VEE-Rep-Cre (simultaneous transduction) (FIG. 9F),or VEERep-Cre, incubation of cells for 4 hr prior to transduction withVLPs containing VEE-Rep-GFP (FIG. 9G).

FIGS. 10A and 10B show a schematic representation of an alphavirusreplicon having two different genes (encoding either HLA-DR1 or CD80)the expression of which can occur in the same cell (FIG. 10A). Images ofcells expressing both proteins following transduction with a VLP havinga replicon shown in FIG. 10A are provided in FIG. 10B, where HLA-DR1expression is visualized using immunospecific labeling with FITC (green)and CD80 is visualized with immunospecific labeling with phycoerythrin(red); a merged image is also shown to illustrate coexpression in thesame cells.

FIG. 11 provides a schematic representation of pDest472-VEE-MCS.

FIG. 12 provides a schematic representation of pDest472-VEE.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Various alphavirus-based expression vectors for transgene expression intarget cells have been described (Xiong C., et al., 1989, Science1188-91; and Bredenbeek P. et al., 1993, J. Virol. 6439-46). For safetyconsiderations these expression systems usually comprise two plasmids.One plasmid contains the coding sequence of the viral replicon (i.e.,non-structural proteins) and an internal promoter and transgene codingregion, while the second plasmid encodes the viral structural genes.These plasmids are used to generate mRNA in vitro, which is thenelectroporated into host cells to generate one-round infectious virusparticles. These viral particles are then used to infect target cellsfor transgene expression. These particles raise safety concerns,however, because recombination between the sequence elements encodingthe non-structural and the structural viral elements can yieldreplication-competent alphavirus particles having the ability to mediatea significant cytopathic effect in vivo.

A possible solution to this problem is to use unrelated VLPs to deliveralphavirus replicons to the cytoplasm of mammalian cells where they canreplicate autonomously and express genes of interest without any nuclearinvolvement. These VLPs can be produced using three vectors. The firstvector comprises the coding sequence for the alphavirus replicon underthe control of a mammalian promoter (e.g., CMV), a retroviral-specificRNA packaging signal, and a gene or polynucleotide of interest. The genemay express a protein with therapeutic or research applications, or ashRNA or other regulatory nucleic acid. The second vector comprisesretroviral Gag. The third vector would provide the suitable envelopeglycoprotein for infection of target cells.

Upon co-transfection into an appropriate packing cell line, RNAmolecules transcribed from the cellular promoter present in the firstvector will be packaged into VLPs produced from the second vector. TheseVLPs can deliver the alphavirus-based replicon to a target cell based onthe envelope glycoprotein present in the VLPs. Once inside the cell, thehost translational machinery will translate the introduced alphavirusRNA and produce alphavirus replication proteins, which will in turnamplify the RNA and express the gene or polynucleotide of interest.Mutant replicons such as the one described above can greatly prolong theduration of expression with minimal impact on the host cell. Moreover,DNA encoding genes for alphavirus structural elements will be absent inthe target cell, so the safety of the proposed system is greatlyenhanced.

Described herein are compositions relating to VLPs and methods formaking and using the described VLPs. The described compositions includeVLPs, and vectors and cells used to produce the described VLPs. Therelated methods described herein relate to methods of producing theVLPs, methods of transducing cells using the VLPs, and methods ofproducing a protein or polynucleotide of interest in a target cell usingthe VLPs described herein. Also described are alphavirus-based repliconsthat allow for expression of proteins or polynucleotides of interest ina target cell without the risk of viral infection.

Definitions

When the terms “one,” “a,” or “an” are used in this disclosure, theymean “at least one” or “one or more,” unless otherwise indicated.

The term “fusogenic protein” as used herein is meant to refer to aprotein that can induce the fusion of the plasma membrane derivedenvelope of the VLP to the membrane of the recipient cell.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression or production of an antibody orantigen-binding fragment thereof may be within the cytoplasm of thecell, or into the extracellular milieu such as the growth medium of acell culture.

“Polynucleotide,” synonymously referred to as “nucleic acid molecule,”“nucleotides” or “nucleic acids,” refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short nucleic acidchains, often referred to as oligonucleotides.

“Replicon” as used herein refers to a polynucleotide having the geneticelements necessary to facilitate replication of its sequence and whilealso being capable of undergoing translation.

“Virus-like particle” (VLP), as used herein, refers to a structureresembling a virus particle. In preferred embodiments, a VLP contains atleast one fusogenic protein displayed on the surface of the particle. Avirus-like particle in accordance with the invention lacks all or partof the replicative components of the viral genome. Typically, avirus-like particle in accordance with the invention does not carrygenetic information encoding for the proteins of the virus-likeparticle.

Vectors

Described herein are vectors for use in producing VLPs carrying analphavirus-based replicon that does not encode alphavirus structuralproteins. To produce VLPS of this sort, several components may beproduced by transfecting or nucleofecting one or more vectors encodingthese components into a cell line for in vitro production. In someembodiments, these components are encoded by separate vectors to reducethe likelihood that the resulting VLP will be replication competent. Forexample, a multi-plasmid system may be used where one plasmid encodesthe genetic material, such as an RNA polynucleotide encoding Sindbisvirus or VEEV nonstructural proteins, to be packaged by the VLP; anotherencodes the structural proteins of the VLP, such as gag; and anotherplasmid encodes a fusion protein, such as VSV-G, to facilitate fusion ofthe VLP to the membrane of a target cell.

The vectors encoding the genetic material to be packaged by a host cellcan take a variety of forms, such as selectable or inducible plasmids,but generally have some common characteristics. For example, vectorsencoding an RNA alphavirus-based replicon described herein may include apromoter sequence, a retroviral packaging signal sequence, translationinitiation sequences, nonstructural alphavirus proteins, a cloning sitefor inserting a gene or polynucleotide of interest, an inserted gene orpolynucleotide of interest, a 3′ untranslated region, and apoly-adenosine tail segment.

In some embodiments the described vectors include a promoter elementthat allows for segments of the vector to be transcribed by a host cell.In some embodiments the vector sequence may be transcribed into RNA tobe packaged into a VLP. In most embodiments of the described vectors,the promoter sequence will be located upstream of all of thetranslatable elements included within the vector (see for example, FIG.1(a) illustrating the location of the cytomegalovirus promoter “pCMV”).In some embodiments described herein the promoter sequence will bederived from a virus, such as cytomegalovirus (CMV), or simian virus 40(SV40). Numerous other promoter sequences are well known to thoseskilled in the art and their use with the vectors described herein wouldbe apparent based on the description provided.

In some embodiments the described vectors encoding the genetic materialto be packaged by a host cell can include a polynucleotide sequence thatencodes a retroviral packaging signal sequence (also known as a psi (ψ)element) to allow one or two copies of the RNA sequence transcribed fromthe vector to be packaged into a VLP particle formed in a host cell.Most, if not all, retroviruses have a packaging sequence of this nature,thus these sequences, and their incorporation into the describedvectors, will be readily apparent to those skilled in the art. In someembodiments the vectors described herein include a polynucleotidesequence that encodes a retroviral packaging sequence derived from Roussarcoma virus, Moloney murine leukemia virus, simian immunodeficiencyvirus (SIV), HIV, human T-lymphotropic virus, and the like. In aparticular embodiment, the retroviral packaging sequence is derived fromRous sarcoma virus. Alternatively, the retroviral packaging sequence isderived from murine leukemia virus.

Another aspect of the vectors encoding the genetic material to bepackaged by a host cell described herein are translation initiationsequences, which allow the RNA sequence encoded by the vector to betranslated in a host cell. In some embodiments the described translationinitiation sequences may be capable of allowing for expression ofalphavirus-based nonstructural proteins, which can replicate the RNAsequence carried by the described VLPs once it is delivered to the hostcell. In some embodiments, the described translation initiationsequences may be capable of allowing for expression of a gene ofinterest. In some embodiments the translation initiation sequence mayallow for the gene of interest to be translated by host cell translationcomplexes. In some embodiments the translation initiation sequencesdescribed herein may be derived from an alphavirus, such as Sindbisvirus or VEEV. In other embodiments the translation initiation sequencesmay be derived from other genes, such as virus genes known to havetranslation initiation sequences capable of initiating translation of anRNA sequence by mammalian translation complexes. Alternatively, thetranslation initiation sequences may be derived from other genes, suchas the native translation initiation sequence of the gene of interestinserted into the described alphavirus replicon. In some embodiments thetranslation initiation sequences described herein may be located at morethan one location in the packaged RNA molecule, and thus may be encodedone or more times by the described vectors. For example, it may bedesirable to translate the described Sindbis or VEEV nonstructuralproteins separately from a gene of interest encoded by the package RNAmolecule. In such an instance, both the polynucleotide(s) encoding thenonstructural proteins and the polynucleotide encoding the protein ofinterest will have separate translation initiation sequences located 5′of their position in the vector and packaged RNA. Based on thisdescription, those skilled in the art will understand that a variety oftranslation initiation sequences capable of promoting the translation ofRNA in a mammalian cell may be incorporated to the describedVLP-packaged RNAs described herein.

The vectors encoding genetic material to be packaged by a host cell mayalso include polynucleotides that encode nonstructural alphavirusproteins, such as nonstructural proteins from Sindbis virus or VEEV. Forexample, in some embodiments the described vectors may includepolynucleotides that encode one or more Sindbis virus nonstructuralproteins. In some embodiments the described vectors may includepolynucleotides that encode one or more VEEV nonstructural proteins. Insome embodiments described vectors may include polynucleotides thatencode the Sindbis virus or VEEV nonstructural protein NSP1. In someembodiments described vectors may include polynucleotides that encodethe Sindbis virus or VEEV nonstructural protein NSP2. In someembodiments described vectors may include polynucleotides that encodethe Sindbis virus or VEEV nonstructural protein NSP3. In someembodiments described vectors may include polynucleotides that encodethe Sindbis virus or VEEV nonstructural protein NSP4. In someembodiments described vectors may include polynucleotides that encodethe Sindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, andNSP4. In some embodiments the polynucleotide of the described vectorthat encodes the alphavirus nonstructural proteins will be derived fromthe corresponding genomic sequence of an alphavirus genome, such as thatof Sindbis virus or VEEV. In some embodiments, the polynucleotidesencoding the alphavirus nonstructural proteins are void of anypolynucleotides that encode the alphavirus structural proteins,regardless of whether the structural proteins are from the same or adifferent alphavirus than the nonstructural protein sequences present.

The vector described herein for incorporating genetic material to bepackaged by a host cell may also contain a polynucleotide of interestthat may be expressed in a host cell transduced by a VLP carrying thegenetic material encoded by the vector. In some embodiments thedescribed vectors may encode an RNA polynucleotide sequence to bepackaged into a VLP, which can then be delivered to a host cell byVLP-mediated transduction of the cell. Once the RNA polynucleotidesequence has been delivered to the target cell a polynucleotide ofinterest encoded by the RNA may provide for expression of a protein ofinterest. Accordingly, the vectors described herein are designed toencode an RNA for packaging into a VLP that can express a gene ofinterest once inside a target cell. Therefore, in some embodiments thedescribed vectors will include a polynucleotide sequence of interest. Insome embodiments of the described vector, the polynucleotide sequence ofinterest may encode a protein of interest. For example, thepolynucleotide sequence of interest may encode GFP in some embodimentsand serve a detectable marker of viral transduction of a target cell. Inanother embodiment, the polynucleotide sequence of interest may encode afunctional version of a protein endogenous to the target cell. Inanother embodiment, the polynucleotide sequence of interest may encode afunctional version of a protein endogenous to the target subject. Inanother embodiment, the polynucleotide sequence of interest may encode aprotein that is foreign to the target cell. In another embodiment, thepolynucleotide sequence of interest may encode a protein that is foreignto the target subject. In some embodiments the polynucleotide sequenceof interest may encode a protein capable of having a therapeutic effecton a target cell. In some embodiments the polynucleotide sequence ofinterest may encode a protein capable of having a therapeutic effect ona target subject. In an alternative embodiment the polynucleotidesequence of interest may server as an interfering RNA molecule andfunction to regulate endogenous gene expression in a host cell. Forexample, in some embodiments the polynucleotide sequence of interest maycomprise a sequence that provides for the formation of an RNA hairpinloop to initiate RNA interference. In addition, the polynucleotide ofinterest could be a positive or negative sense strand of RNA that can betranscribed by the RNA-dependent RNA polymerase complex formed by thealphavirus nonstructural proteins encoded by the packaged RNA molecule.Since this RNA-dependent RNA polymerase can transcribe RNA in thepositive-sense and negative-sense directions, an interfering RNAsequence, such as a miRNA or shRNA, may be inserted into the packagedRNA replicon and can be designed to encode an interfering polynucleotidein either direction. Those of skill in the art will appreciate thetherapeutic characteristic of this aspect of the described transductionsystem, as it can allow for expression of selected proteins in asubject. In accordance with this aspect of the described vector, acloning site having one or more restriction endonuclease sites may alsobe included in the vector, to facilitate insertion of a polynucleotidesequence of interest.

Another vector useful in the production of the VLPs described herein isa vector that encodes a virus structural protein. One such class ofproteins is the retroviral group-specific antigen (gag) protein. The gagprotein is the core structural protein of retroviruses and, in someinstances, is capable of forming enveloped virus cores when expressed ineukaryotic cells. This property makes gag proteins particularly usefulin the production of VLPs, because they can form the basic structuralaspect of the particle and allow for packaging of RNA associated with aretroviral packaging signal sequence. Accordingly, described herein arevectors that include a polynucleotide that encodes a retroviral gagprotein. In some embodiments, the described vectors include apolynucleotide that encodes a retroviral gag protein and a promoterpolynucleotide sequence that allows for the gag gene sequence to betranscribed into mRNA by host cell RNA polymerase. In one embodiment,the promoter polynucleotide sequence is derived from a virus, such asSV40 or CMV. In some embodiments, the vector will further include apolynucleotide that encodes a protein of interest. Those skilled in therelevant art will understand that a polynucleotide sequence of a gagprotein from any retrovirus may be used to produce the vectors and VLPsdescribed herein. In some embodiments the polynucleotide sequenceencoding the gag protein may be derived from Rous sarcoma virus. In someembodiments the polynucleotide sequence encoding the gag protein may bederived from murine leukemia virus. In some embodiments thepolynucleotide sequence encoding the gag protein may be derived fromSIV. In some embodiments the polynucleotide sequence encoding the gagprotein may be derived from human T-lymphotropic virus.

Another vector useful in the production of the VLPs described herein isa vector that encodes a protein to mediate fusion between the VLPenvelope and a host cell. A class of proteins suitable for this purposeis viral fusion proteins, which facilitate virus infection of cells byallowing an enveloped virus to fuse its membrane with that of a hostcell. Many of viral fusion proteins also have known, or suspected,cellular receptor proteins that may allow for targeting of selected celltypes, or in cases of more ubiquitous receptors, such as sialic acid forinfluenza virus, more generalized targeting may be desired. In someinstances, viral fusion proteins work in conjunction with viralattachment proteins, ligands for cellular receptor, a receptor for acell ligand, or accessory proteins, thus proteins of this sort may alsobe encoded by the described vectors, in addition to, or also by, thevector encoding a viral fusion protein. Alternatively, in someembodiments a viral fusion protein from one virus may be encoded by thedescribed vector along with a viral attachment protein of another virus,a ligand of a cellular receptor, a receptor of a cell ligand, or anaccessory protein to facilitate, or direct, targeting of a VLP to adesired cell type. In some embodiments the viral fusion protein, viralattachment protein, ligand of a cellular receptor, receptor of a cellligand, or accessory protein will be a type-I membrane protein, whichwill allow the extracellular domain of the protein to be orientedextracellularly when present on the cell surface. This will also allowthe fusion protein to be correctly oriented following budding of a VLPfrom a packaging cell. Expression of such proteins in a cell willtypically result in the cell surface being coated with the proteins, sothat budding of a VLP from any part of the cell membrane will providethe VLP with some amount of the protein(s) on its surface. In someembodiments, the described vectors include a polynucleotide that encodesa viral fusion protein and a promoter polynucleotide sequence thatallows for the fusion protein gene sequence to be translated into mRNAby host cell RNA polymerase. In one embodiment, the promoterpolynucleotide sequence is derived from a virus, such as SV40 or CMV. Insome embodiments, the described vectors include a polynucleotide thatencodes a viral attachment protein and a promoter polynucleotidesequence that allows for the attachment protein gene sequence to betranslated into mRNA by host cell RNA polymerase. In one embodiment, thepromoter polynucleotide sequence is derived from a virus, such as SV40or CMV. In some embodiments the vectors described herein include apolynucleotide that encodes a vesicular stomatitis virus G protein. Insome embodiments the vectors described herein include a polynucleotidethat encodes the influenza hemaglutinin protein. In some embodiments thevectors described herein include a polynucleotide that encodes theinfluenza neuraminidase protein. In some embodiments the vectorsdescribed herein include a polynucleotide that encodes the respiratorysyncytial virus fusion protein. In some embodiments the vectorsdescribed herein include a polynucleotide that encodes the rotavirus VP7protein. Other such fusion proteins will be apparent to those skilled inthe art based on desired tropism or cell target of the associated virus.

Cells Expressing the Described Vectors

Provided herein are cells comprising the vectors described to produceVLPs. These cells may be used to produce the VLPs described herein bytranscribing or expressing the polynucleotides of the vectors. Forinstance, a mammalian cell transfected with a vector having apolynucleotide sequence encoding an alphavirus RNA construct having agene or polynucleotide of interest and a packaging signal, a vectorencoding a retroviral gag protein, and a vector encoding a viral fusionprotein could produce a VLP having the expressed viral fusion protein onits surface with one or two copies of the encoded alphavirus RNAconstruct housed inside the VLP. Furthermore, because none of thesevectors encode alphavirus structural proteins the possibility ofcreating an infectious virus is substantially reduced compared tosystems that do include alphavirus structural proteins.

The described cells may be any eukaryotic cell capable of transcribing,and where necessary (such as in the case of the gag and fusionproteins), translating the polynucleotides of the described vectors. Thecells will likely be mammalian cells in many embodiments. For example,rodent cells, such as murine, hamster (CHO or BHK-21), or rat cellscould be used to express the described vectors; canine cells, such asMadin Darby canine kidney cells, could be used to express the describedvectors; primate cells, such as vero cells, could be used to express thedescribed vectors; and human cells, such as HEK293T cells (humankidney), Hep-2 cells (human airway), Caco-2 (intestine), HeLa(epithelium), and other such cell lines known in the art, could be usedto express the described vectors. In some embodiments the describedcells can be transfected and selected, using standard transfection andselection methods known in the art, to stably comprise one or more ofthe described vectors.

In some embodiments the cell lines described herein will contain avector comprising a polynucleotide sequence encoding an alphavirusreplicon wherein the alphavirus replicon encodes a protein of interest,a vector comprising a polynucleotide sequence encoding a gag protein,and a vector comprising a polynucleotide sequence encoding aheterologous fusogenic envelope protein, wherein neither the vectors northe cell contain a gene encoding an alphavirus structural protein. Insome embodiments the alphavirus replicon may be derived from Sindbisvirus or VEEV. In some embodiments the alphavirus replicon may havepolynucleotide sequences that encode Sindbis virus or VEEV nonstructuralproteins NSP1, NSP2, NSP3, NSP4, and a retroviral packaging signal. Insome embodiments the retroviral packaging signal may be derived fromeither Rous sarcoma virus or murine leukemia virus. In some embodimentsthe polynucleotide sequence encoding the gag protein is derived fromRous sarcoma virus. In some embodiments the polynucleotide sequenceencoding the heterologous fusogenic envelope protein encodes VSV-G.

Virus-Like Particles

VLPs produced using the vectors and cells are also described herein. TheVLPs described herein will have four general characteristics: they willcomprise one or two RNA molecules encoding an alphavirus replicon, andoptionally a protein of interest; they will have a viral core comprisinga retroviral gag protein, or, in some embodiments, a gag fusion protein;they will have a surface protein to facilitate fusion with a cell, andthey will not contain a polynucleotide that encodes an alphavirusstructural protein.

The VLPs described herein will be useful in transducing cells in orderto express a protein of interest therein. Accordingly, the describedVLPs may incorporate one or two alphavirus-based RNA polynucleotidescapable of encoding a protein of interest. To facilitate translation ofthe RNA sequence some embodiments of the described packaged RNA mayinclude translation initiation sequences as described herein. In someembodiments the RNA sequence incorporated into the VLP will include aretroviral packaging sequence that will facilitate inclusion of the RNAinto a forming VLP. In some embodiments the retroviral packagingsequence is derived from Rous sarcoma virus, Moloney murine leukemiavirus, simian immunodeficiency virus (SIV), HIV, human T-lymphotropicvirus, and the like. In a particular embodiment, the retroviralpackaging sequence is derived from Rous sarcoma virus. Alternatively,the retroviral packaging sequence may be derived from murine leukemiavirus. In addition, the RNA sequences included in the VLP may be capableof encoding nonstructural alphavirus proteins. For example, in someembodiments the packaged RNA may encode one or more Sindbis virus orVEEV nonstructural proteins. In some embodiments the packaged RNA mayencode the Sindbis virus or VEEV nonstructural protein NSP1. In someembodiments the packaged RNA may encode the Sindbis virus or VEEVnonstructural protein NSP2. In some embodiments the packaged RNA mayencode the Sindbis virus or VEEV nonstructural protein NSP3. In someembodiments the packaged RNA may encode the Sindbis virus or VEEVnonstructural protein NSP4. In some embodiments the packaged RNA mayencode the Sindbis virus or VEEV nonstructural proteins NSP1, NSP2,NSP3, and NSP4. The packaged RNA may also include the polynucleotidesequence of a protein of interest. For example, the polynucleotidesequence of interest may encode GFP in some embodiments and serve adetectable marker of viral transduction of a target cell. In anotherembodiment, the polynucleotide sequence of interest may encode afunctional version of a protein endogenous to the target cell. Inanother embodiment, the polynucleotide sequence of interest may encode afunctional version of a protein endogenous to the target subject. Inanother embodiment, the polynucleotide sequence of interest may encode aprotein that is foreign to the target cell. In another embodiment, thepolynucleotide sequence of interest may encode a protein that is foreignto the target subject. In some embodiments the polynucleotide sequenceof interest may encode a protein capable of having a therapeutic effecton a target cell. In some embodiments the polynucleotide sequence ofinterest may encode a protein capable of having a therapeutic effect ona target subject. Those of skill in the art will appreciate thetherapeutic characteristic of this aspect of the described VLPs, as theycan allow for expression of selected proteins in a cell or subject.

The described VLPs may also comprise a viral gag protein to provide aviral core structure to the particle. The gag protein is the corestructural protein of retroviruses and, in some instances, is capable offorming enveloped virus cores when expressed in eukaryotic cells. Thisproperty makes gag proteins particularly useful in the production ofVLPs, because they can form the basic structural aspect of the particleand allow for packaging of RNA associated with a retroviral packagingsignal sequence. Those skilled in the relevant art will understand thata gag protein from any retrovirus may be used to produce the vectors andVLPs described herein. In some embodiments the gag protein may bederived from Rous sarcoma virus. In some embodiments the gag protein maybe derived from murine leukemia virus. In alternative embodiments thegag protein may be derived from SIV, HIV, human T-lymphotropic virus, orsimilar retrovirus.

Another component of the VLPs described herein is a protein to mediatefusion between the VLP envelope and a host cell. A class of proteinssuitable for this purpose is viral fusion proteins, which facilitatevirus infection of cells by allowing an enveloped virus to fuse itsmembrane with that of a host cell. Many of viral fusion proteins alsohave known, or suspected, cellular receptor proteins that may allow fortargeting of selected cell types, or in cases of more ubiquitousreceptors, such as sialic acid for influenza virus, more generalizedtargeting may be achieved. In some instances, viral fusion proteins maywork in conjunction with viral attachment proteins, ligands of cellularreceptors, receptors of cellular ligands, or accessory proteins, thusproteins of this sort may also be present on the VLP surface in additionto a viral fusion protein. Alternatively, in some embodiments thedescribed VLPs may have a viral fusion protein from one virus and aviral attachment protein of another virus, a ligand of a cellularreceptor, a receptor of a cellular ligand, or an accessory protein tofacilitate, or direct, targeting of a VLP to a desired cell type.Similarly, the described VLPs may be produced to have more than onefusion protein in the VLP envelope, as this may facilitate fusion to aselect variety of cell types. In some embodiments the VLP surfaceprotein(s) will be a type-I membrane protein, which will allow theextracellular domain of the protein to be oriented extracellularly whenpresent on the cell surface. This will also allow the fusion protein tobe correctly oriented following budding of a VLP from a packaging cell.Expression of such proteins in a cell will typically result in the cellsurface being coated with the proteins, so that budding of a VLP fromany part of the cell membrane will provide the VLP with some amount ofthe fusion protein on its surface. In some embodiments the VLPsdescribed herein include a vesicular stomatitis virus G protein (VSV-G)to mediate cell fusion. In some embodiments the VLPs described hereininclude an influenza hemaglutinin protein to mediate cell fusion. Insome embodiments the VLPs described herein include an influenzaneuraminidase protein to facilitate cell fusion. In some embodiments theVLPs described herein include respiratory syncytial virus fusionprotein. In some embodiments the VLPs described herein include therotavirus VP7 protein. Other such fusion proteins will be apparent tothose skilled in the art based on desired tropism or cell target of theassociated virus.

The VLPs described herein may comprise an alphavirus replicon, whereinthe alphavirus replicon includes a polynucleotide of interest or encodesa protein of interest, retroviral gag protein, and heterologousfusogenic envelope protein; wherein the VLP does not contain analphavirus structural protein gene. In some embodiments the alphavirusreplicon of the VLP is derived from Sindbis virus or VEEV. For example,the VLPs described herein may have an alphavirus replicon encodingSindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, and NSP4.In some embodiments the retroviral packaging signal associated with thepackaged RNA in the described VLPs is derived from either Rous sarcomavirus or murine leukemia virus. Based on this description, those skilledin the art will readily understand that the described VLPs may bemodified to incorporate aspects of viruses that may facilitate VLPstability, RNA packaging, or cell entry. Such modifications should beunderstood to be within the scope of the disclosures provided herein.

Methods of Producing the Described VLPs

The VLPs described herein may be produced in a variety of ways, as willbe apparent to those skilled in the art based on the provideddisclosure. The commonality to these various methods is the expressionof the described vectors in a cell capable of expressing the necessaryproteins (gag and a fusion protein) and producing the alphavirus-basedRNA replicon. Accordingly, a method of producing a VLP described hereinmay include co-transforming, transfecting, or nucleofecting a eukaryoticcell with a vector comprising a polynucleotide sequence encoding analphavirus replicon, wherein the alphavirus replicon includes apolynucleotide of interest or encodes a protein of interest; a vectorcomprising a polynucleotide sequence encoding a retroviral gag protein;and a vector comprising a polynucleotide sequence encoding aheterologous fusogenic envelope protein; and culturing theco-transformed cell under conditions suitable to cause each vector toproduce its encoded product, thereby producing a virus-like particle. Insome embodiments the polynucleotide sequence encoding the alphavirusreplicon may be derived from Sindbis virus or VEEV. In some embodimentsthe alphavirus replicon may have polynucleotide sequences that encodeSindbis virus or VEEV nonstructural proteins NSP1, NSP2, NSP3, NSP4, anda retroviral packaging signal. In some embodiments the retroviralpackaging signal may be derived from either Rous sarcoma virus or murineleukemia virus. In some embodiments the polynucleotide sequence encodingthe gag protein is derived from Rous sarcoma virus. In some embodimentsthe polynucleotide sequence encoding the heterologous fusogenic envelopeprotein encodes VSV-G.

Compositions and Methods of Administration

Described herein are compositions comprising at least one described VLPand a pharmaceutically acceptable carrier. Such compositions are useful,for example, for administration to subjects in need of expression of anexogenous protein or increased expression of a protein normally found inthose of the same species as the subject. The compositions may beformulated as any of various preparations that are known and suitable inthe art, including those described and exemplified herein. In someembodiments, the compositions are aqueous formulations. Aqueoussolutions may be prepared by admixing the VLPs in water or suitablephysiologic buffer, and optionally adding suitable colorants, flavors,preservatives, stabilizing and thickening agents and the like asdesired. Aqueous suspensions may also be made by dispersing the VLPs inwater or physiologic buffer with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

The compositions may be formulated for injection into a subject. Forinjection, the compositions described may be formulated in aqueoussolutions such as water or in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Thesolution may contain one or more formulatory agents such as suspending,stabilizing or dispersing agents. Injection formulations may also beprepared as solid form preparations which are intended to be converted,shortly before use, to liquid form preparations suitable for injection,for example, by constitution with a suitable vehicle, such as sterilewater, saline solution, or alcohol, before use.

The compositions may be formulated for aerosolized delivery to asubject. For aerosol delivery, the compositions described may beformulated in aqueous solutions such as water or in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. The solution may contain one or moreformulatory agents such as suspending, stabilizing or dispersing agents.

The compositions may be formulated in sustained release vehicles ordepot preparations. Such long-acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Liposomes and emulsions are well-known examples of deliveryvehicles suitable for use as carriers for hydrophobic drugs.

The following examples are provided for illustrative purposes and aremeant to enhance, not limit, the preceding disclosure.

EXAMPLE 1 Production of an Alphavirus-Based Gene Expression System

An alphavirus gene expression system was designed to allow forVLP-mediated delivery an exogenous gene of interest (GOI) or protein ofinterest (POI) to a target cell with low risk of causing cytopathicviral infection. The expression system was designed using three vectors,which can be expressed in a packaging cell line to produce a transducingVLP. One vector codes for the alphavirus-based expression construct,another vector codes for a retroviral gag protein to facilitate VLPformation, and a third vector codes for a fusion protein to mediate VLPfusion to the host cell. In addition, the system was constructed to workwithout the need for alphavirus structural proteins being present.

To accomplish this, an alphavirus-based DNA plasmid was produced havinga cytomegalovirus promoter (CMV); followed by a retroviral packagingsignal of respective retroviral packaging protein GAG; followed by aSindbis or VEE virus genes encoding nonstructural proteins NSP1, NSP2,NSP3, and NSP4; and finally, one or more subgenomic promoter (SGP; apromoter for virus-encoded RNA-dependent RNA polymerase, resulting inthe formation of mRNA) to drive expression of a of a gene of interest(GOI), consisting of a recombinant polynucleotide, and inserted into amultiple cloning site; a 3′ untranslated region (URT); and a polyA tail.FIG. 2 shows an example of such an alphavirus-based DNA plasmid. Inanother version of this expression vector, the retroviral packagingsignal (GAG) is omitted.

Another plasmid was constructed to encode a retroviral gag protein and asecond, optional protein of interest (POI). A third plasmid wasconstructed to provide expression of a VSV-G viral fusion protein. Aschematic of an embodiment of these plasmids is provided in FIG. 1A-1C,respectively. FIG. 1A shows pCMV-Sin Rep-POI-2, FIG. 1B showspGAG-POI-1, and FIG. 1C shows pEnv for VSV-G.

Once constructed the plasmids were tested for the ability to produceVLPs carrying a Sindbis virus replicon having a gene of interest. Forthese experiments, green fluorescent protein (GFP) was used as the geneof interest in order to facilitate detection of delivery andintracellular expression of the gene. To produce VLPs, each of the threeplasmids described above were transfected into baby hamster kidney(BHK-21) cells using a standard nucleofection procedure with an Amaxasystem according to manufacturer instructions (Lonza) (FIG. 3).

Briefly, the BHK-21 cells at 3×10⁶ were re-suspended in 100 μlnucleofection solution L (Amaxa) and transferred to tube containing 4.5μg of plasmid coding for GAG, 3 plasmid coding for VSV-G glycoproteinand 100 nanograms of plasmid coding for Sindbis alphavirus replicon or2.5 micrograms for VEE replicon (in total volume of 10 μl). The mixtureof cells and plasmids was transferred to nucleofection cuvette andnucleofected using Amaxa nucleofector II apparatus using settings forBHK-21. The nucleofected cells were resuspended in 500 μl of completedculture medium and transferred to tissue culture plate containingculture medium solution and incubated at 37° C. for period of 72-96 hror for 72 hr at 32° C. After this time supernatants consisting of VLPsand encapsidated alphavirus replicon was clarified by centrifugation at3000 RPM/10 min at 4° C., filtered by 0.45 um filter and exposed to 10units of DNAse I (turbo™-DNAse (Ambion)) for 30 min at RT. ProcessedVLPs were stored at 4° C. or frozen on dry ice and transferred to −80°C. As a negative control (fusion-defective VLPs), BHK-21 cells were alsonucleofected with only the pCMV-Sin Rep-POI-2 or VEEV-Rep-POI andpGAG-POI-1 plasmids, but not the pEnvelope plasmid encoding VSV-G.Following transfection, the cells were incubated for 48-72 hours intissue culture medium under normal growth conditions to allow forplasmid-driven production of VLPs. Once the transfected cells werefinished incubating, the tissue culture supernatant, which shouldcontain any produced VLPs, was collected. The collected cellsupernatants were then added to cultured BHK-21 cells to determine ifthe cells could be successfully transduced with GFP. As shown in FIG. 4,cell supernatants collected from BHK-21 cells transfected with all threeplasmids resulted in robust GFP expression when exposed to untransfectedBHK-21 cells (FIG. 4A). Conversely, no GFP expression was observed foruntransfected BHK-21 cells incubated with cell supernatants collectedfrom BHK-21 cells transfected with only the pCMV-Sin Rep-POI-2 andpGAG-POI-1 plasmids (FIG. 4B). Similar experiments were also conductedusing human embryonic kidney (HEK293T) cells to demonstrate that theconstructed VLPs could transduce human cells (FIG. 4C). Furthermore, theconstructed VLPs can also be stored at 4° C. for at least 30 dayswithout losing the ability to transduce cells (FIG. 5A-5D).

Experiments were also conducted to assess the ability of VEEV-basedalpha virus replicon to express protein in cells. For these studiesBHK-21 cells were transduced with VLPs having a Gaussia luciferase geneinserted into a VEEV replicon. Following transduction, cell supernatantsmonitored for expression of luciferase protein. As shown in FIG. 6, highamounts of luciferase were detected in the supernatants of cellstransduced with the VEEV replicon having the Gaussia luciferase gene(FIG. 6A, condition 4), relative to control VEEV replicons without anexogenous gene (condition 1), or with a gene encoding GFP (conditions 2and 3). Additionally, expression of the luciferase protein increasedrapidly after transduction (FIG. 6B). Similar results were also observedin the context of delivering functional cre recombinase (red cells) tocells engineered to express GFP in the absence of cre recombinase (FIG.7B).

Cells were transduced in parallel with either Sindbis-based VLPsencoding GFP or VEEV-based VLPs encoding GFP. As shown in FIG. 8, bothalphavirus-based VLPs caused robust GFP expression, while the cellstransduced with VEEV-based VLPs were observed to have the higherexpression levels (FIG. 8).

EXAMPLE 2 miRNA Expressed by a VEE Alphavirus Replicon Inhibits ProteinProduction

Experiments were conducted to assess whether an alphavirus repliconencoding an miRNA sequence could inhibit protein production. To assessthis, BHK-21 cells were transduced with a VLP replicon encoding GFP,miRNA specific for GFP, or cre recombinase (FIG. 9). (See the vectorsshown in FIGS. 11 and 12, and SEQ ID NO:1 in Table 1, below.) GFPexpression was noticeable reduced when cells were first transduced witha replicon encoding miRNA for GFP and then subsequently transduced witha replicon encoding GFP (4 hours after the intial transduction), ascompared to cells transduced with only a replicon encoding GFP (compareFIG. 9A with FIG. 9D). Conversely, when cells were first transduced witha replicon encoding cre recombinase and then subsequently transducedwith a replicon encoding GFP (4 hours after the initial transduction),no significant reduction in GFP expression was apparent (FIG. 9G).

EXAMPLE 3 Expression of Multiple Proteins in Target Cells Transducedwith VEE VLPs

To assess whether an alphavirus replicon can express two separateproteins in the same cell, experiments were performed using a VEEreplicon having HLA-DR1 under the control of one subgenomic promoter andCD80 under a another subgenomic promotor (FIG. 10A). Followingproduction of VLPs having the described VEE replicon, cells weretransduced and examined for expression of both proteins. As shown inFIG. 10B, transduced cells were able to express both proteins(immunolabled HLA-DR1 is shown in green (FITC) and CD80 shown in red(phycoerythrin).

TABLE 1 SEQ ID NO: 1     1gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg    61ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg   121cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc   181ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt   241gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata   301tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc   361cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc   421attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt   481atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt   541atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca   601tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg   661actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc   721aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg   781gtaggcgtgt acggtgggag gtctatataa gcagagctcg tatggacata ttgtcgttag   841aacgcggcta caattaatac ataaccttat gtatcataca catacgattt aggggacact   901atagattgac ggcgtagtac acactattga atcaaacagc cgaccaattg cactaccatc   961acaatggaga agccagtagt aaacgtagac gtagaccccc agagtccgtt tgtcgtgcaa  1021ctgcaaaaaa gcttcccgca atttgaggta gtagcacagc aggtcactcc aaatgaccat  1081gctaatgcca gagcattttc gcatctggcc agtaaactaa tcgagctgga ggttcctacc  1141acagcgacga tcttggacat aggcagcgca ccggctcgta gaatgttttc cgagcaccag  1201tatcattgtg tctgccccat gcgtagtcca gaagacccgg accgcatgat gaaatacgcc  1261agtaaactgg cggaaaaagc gtgcaagatt acaaacaaga acttgcatga gaagattaag  1321gatctccgga ccgtacttga tacgccggat gctgaaacac catcgctctg ctttcacaac  1381gatgttacct gcaacatgcg tgccgaatat tccgtcatgc aggacgtgta tatcaacgct  1441cccggaacta tctatcatca ggctatgaaa ggcgtgcgga ccctgtactg gattggcttc  1501gacaccaccc agttcatgtt ctcggctatg gcaggttcgt accctgcgta caacaccaac  1561tgggccgacg agaaagtcct tgaagcgcgt aacatcggac tttgcagcac aaagctgagt  1621gaaggtagga caggaaaatt gtcgataatg aggaagaagg agttgaagcc cgggtcgcgg  1681gtttatttct ccgtaggatc gacactttat ccagaacaca gagccagctt gcagagctgg  1741catcttccat cggtgttcca cttgaatgga aagcagtcgt acacttgccg ctgtgataca  1801gtggtgagtt gcgaaggcta cgtagtgaag aaaatcacca tcagtcccgg gatcacggga  1861gaaaccgtgg gatacgcggt tacacacaat agcgagggct tcttgctatg caaagttact  1921gacacagtaa aaggagaacg ggtatcgttc cctgtgtgca cgtacatccc ggccaccata  1981tgcgatcaga tgactggtat aatggccacg gatatatcac ctgacgatgc acaaaaactt  2041ctggttgggc tcaaccagcg aattgtcatt aacggtagga ctaacaggaa caccaacacc  2101atgcaaaatt accttctgcc gatcatagca caagggttca gcaaatgggc taaggagcgc  2161aaggatgatc ttgataacga gaaaatgctg ggtactagag aacgcaagct tacgtatggc  2221tgcttgtggg cgtttcgcac taagaaagta cattcgtttt atcgcccacc tggaacgcag  2281acctgcgtaa aagtcccagc ctcttttagc gcttttccca tgtcgtccgt atggacgacc  2341tctttgccca tgtcgctgag gcagaaattg aaactggcat tgcaaccaaa gaaggaggaa  2401aaactgctgc aggtctcgga ggaattagtc atggaggcca aggctgcttt tgaggatgct  2461caggaggaag ccagagcgga gaagctccga gaagcacttc caccattagt ggcagacaaa  2521ggcatcgagg cagccgcaga agttgtctgc gaagtggagg ggctccaggc ggacatcgga  2581gcagcattag ttgaaacccc gcgcggtcac gtaaggataa tacctcaagc aaatgaccgt  2641atgatcggac agtatatcgt tgtctcgcca aactctgtgc tgaagaatgc caaactcgca  2701ccagcgcacc cgctagcaga tcaggttaag atcataacac actccggaag atcaggaagg  2761tacgcggtcg aaccatacga cgctaaagta ctgatgccag caggaggtgc cgtaccatgg  2821ccagaattcc tagcactgag tgagagcgcc acgttagtgt acaacgaaag agagtttgtg  2881aaccgcaaac tataccacat tgccatgcat ggccccgcca agaatacaga agaggagcag  2941tacaaggtta caaaggcaga gcttgcagaa acagagtacg tgtttgacgt ggacaagaag  3001cgttgcgtta agaaggaaga agcctcaggt ctggtcctct cgggagaact gaccaaccct  3061ccctatcatg agctagctct ggagggactg aagacccgac ctgcggtccc gtacaaggtc  3121gaaacaatag gagtgatagg cacaccgggg tcgggcaagt cagctattat caagtcaact  3181gtcacggcac gagatcttgt taccagcgga aagaaagaaa attgtcgcga aattgaggcc  3241gacgtgctaa gactgagggg tatgcagatt acgtcgaaga cagtagattc ggttatgctc  3301aacggatgcc acaaagccgt agaagtgctg tacgttgacg aagcgttcgc gtgccacgca  3361ggagcactac ttgccttgat tgctatcgtc aggccccgca agaaggtagt actatgcgga  3421gaccccatgc aatgcggatt cttcaacatg atgcaactaa aggtacattt caatcaccct  3481gaaaaagaca tatgcaccaa gacattctac aagtatatct cccggcgttg cacacagcca  3541gttacagcta ttgtatcgac actgcattac gatggaaaga tgaaaaccac gaacccgtgc  3601aagaagaaca ttgaaatcga tattacaggg gccacaaagc cgaagccagg ggatatcatc  3661ctgacatgtt tccgcgggtg ggttaagcaa ttgcaaatcg actatcccgg acatgaagta  3721atgacagccg cggcctcaca agggctaacc agaaaaggag tgtatgccgt ccggcaaaaa  3781gtcaatgaaa acccactgta cgcgatcaca tcagagcatg tgaacgtgtt gctcacccgc  3841actgaggaca ggctagtgtg gaaaaccttg cagggcgacc catggattaa gcagcccact  3901aacataccta aaggaaactt tcaggctact atagaggact gggaagctga acacaaggga  3961ataattgctg caataaacag ccccactccc cgtgccaatc cgttcagctg caagaccaac  4021gtttgctggg cgaaagcatt ggaaccgata ctagccacgg ccggtatcgt acttaccggt  4081tgccagtgga gcgaactgtt cccacagttt gcggatgaca aaccacattc ggccatttac  4141gccttagacg taatttgcat taagtttttc ggcatggact tgacaagcgg actgttttct  4201aaacagagca tcccactaac gtaccatccc gccgattcag cgaggccggt agctcattgg  4261gacaacagcc caggaacccg caagtatggg tacgatcacg ccattgccgc cgaactctcc  4321cgtagatttc cggtgttcca gctagctggg aagggcacac aacttgattt gcagacgggg  4381agaaccagag ttatctctgc acagcataac ctggtcccgg tgaaccgcaa tcttcctcac  4441gccttagtcc ccgagtacaa ggagaagcaa cccggcccgg tcaaaaaatt cttgaaccag  4501ttcaaacacc actcagtact tgtggtatca gaggaaaaaa ttgaagctcc ccgtaagaga  4561atcgaatgga tcgccccgat tggcatagcc ggtgcagata agaactacaa cctggctttc  4621gggtttccgc cgcaggcacg gtacgacctg gtgttcatca acattggaac taaatacaga  4681aaccaccact ttcagcagtg cgaagaccat gcggcgacct taaaaaccct ttcgcgttcg  4741gccctgaatt gccttaaccc aggaggcacc ctcgtggtga agtcctatgg ctacgccgac  4801cgcaacagtg aggacgtagt caccgctctt gccagaaagt ttgtcagggt gtctgcagcg  4861agaccagatt gtgtctcaag caatacagaa atgtacctga ttttccgaca actagacaac  4921agccgtacac ggcaattcac cccgcaccat ctgaattgcg tgatttcgtc cgtgtatgag  4981ggtacaagag atggagttgg agccgcgccg tcataccgca ccaaaaggga gaatattgct  5041gactgtcaag aggaagcagt tgtcaacgca gccaatccgc tgggtagacc aggcgaagga  5101gtctgccgtg ccatctataa acgttggccg accagtttta ccgattcagc cacggagaca  5161ggcaccgcaa gaatgactgt gtgcctagga aagaaagtga tccacgcggt cggccctgat  5221ttccggaagc acccagaagc agaagccttg aaattgctac aaaacgccta ccatgcagtg  5281gcagacttag taaatgaaca taacatcaag tctgtcgcca ttccactgct atctacaggc  5341atttacgcag ccggaaaaga ccgccttgaa gtatcactta actgcttgac aaccgcgcta  5401gacagaactg acgcggacgt aaccatctat tgcctggata agaagtggaa ggaaagaatc  5461gacgcggcac tccaacttaa ggagtctgta acagagctga aggatgaaga tatggagatc  5521gacgatgagt tagtatggat tcatccagac agttgcttga agggaagaaa gggattcagt  5581actacaaaag gaaaattgta ttcgtacttc gaaggcacca aattccatca agcagcaaaa  5641gacatggcgg agataaaggt cctgttccct aatgaccagg aaagtaatga acaactgtgt  5701gcctacatat tgggtgagac catggaagca atccgcgaaa agtgcccggt cgaccataac  5761ccgtcgtcta gcccgcccaa aacgttgccg tgcctttgca tgtatgccat gacgccagaa  5821agggtccaca gacttagaag caataacgtc aaagaagtta cagtatgctc ctccaccccc  5881cttcctaagc acaaaattaa gaatgttcag aaggttcagt gcacgaaagt agtcctgttt  5941aatccgcaca ctcccgcatt cgttcccgcc cgtaagtaca tagaagtgcc agaacagcct  6001accgctcctc ctgcacaggc cgaggaggcc cccgaagttg tagcgacacc gtcaccatct  6061acagctgata acacctcgct tgatgtcaca gacatctcac tggatatgga tgacagtagc  6121gaaggctcac ttttttcgag ctttagcgga tcggacaact ctattactag tatggacagt  6181tggtcgtcag gacctagttc actagagata gtagaccgaa ggcaggtggt ggtggctgac  6241gttcatgccg tccaagagcc tgcccctatt ccaccgccaa ggctaaagaa gatggcccgc  6301ctggcagcgg caagaaaaga gcccactcca ccggcaagca atagctctga gtccctccac  6361ctctcttttg gtggggtatc catgtccctc ggatcaattt tcgacggaga gacggcccgc  6421caggcagcgg tacaacccct ggcaacaggc cccacggatg tgcctatgtc tttcggatcg  6481ttttccgacg gagagattga tgagctgagc cgcagagtaa ctgagtccga acccgtcctg  6541tttggatcat ttgaaccggg cgaagtgaac tcaattatat cgtcccgatc agccgtatct  6601tttccactac gcaagcagag acgtagacgc aggagcagga ggactgaata ctgactaacc  6661ggggtaggtg ggtacatatt ttcgacggac acaggccctg ggcacttgca aaagaagtcc  6721gttctgcaga accagcttac agaaccgacc ttggagcgca atgtcctgga aagaattcat  6781gccccggtgc tcgacacgtc gaaagaggaa caactcaaac tcaggtacca gatgatgccc  6841accgaagcca acaaaagtag gtaccagtct cgtaaagtag aaaatcagaa agccataacc  6901actgagcgac tactgtcagg actacgactg tataactctg ccacagatca gccagaatgc  6961tataagatca cctatccgaa accattgtac tccagtagcg taccggcgaa ctactccgat  7021ccacagttcg ctgtagctgt ctgtaacaac tatctgcatg agaactatcc gacagtagca  7081tcttatcaga ttactgacga gtacgatgct tacttggata tggtagacgg gacagtcgcc  7141tgcctggata ctgcaacctt ctgccccgct aagcttagaa gttacccgaa aaaacatgag  7201tatagagccc cgaatatccg cagtgcggtt ccatcagcga tgcagaacac gctacaaaat  7261gtgctcattg ccgcaactaa aagaaattgc aacgtcacgc agatgcgtga actgccaaca  7321ctggactcag cgacattcaa tgtcgaatgc tttcgaaaat atgcatgtaa tgacgagtat  7381tgggaggagt tcgctcggaa gccaattagg attaccactg agtttgtcac cgcatatgta  7441gctagactga aaggccctaa ggccgccgca ctatttgcaa agacgtataa tttggtccca  7501ttgcaagaag tgcctatgga tagattcgtc atggacatga aaagagacgt gaaagttaca  7561ccaggcacga aacacacaga agaaagaccg aaagtacaag tgatacaagc cgcagaaccc  7621ctggcgactg cttacttatg cgggattcac cgggaattag tgcgtaggct tacggccgtc  7681ttgcttccaa acattcacac gctttttgac atgtcggcgg aggattttga tgcaatcata  7741gcagaacact tcaagcaagg cgacccggta ctggagacgg atatcgcatc attcgacaaa  7801agccaagacg acgctatggc gttaaccggt ctgatgatct tggaggacct gggtgtggat  7861caaccactac tcgacttgat cgagtgcgcc tttggagaaa tatcatccac ccatctacct  7921acgggtactc gttttaaatt cggggcgatg atgaaatccg gaatgttcct cacacttttt  7981gtcaacacag ttttgaatgt cgttatcgcc agcagagtac tagaagagcg gcttaaaacg  8041tccagatgtg cagcgttcat tggcgacgac aacatcatac atggagtagt atctgacaaa  8101gaaatggctg agaggtgcgc cacctggctc aacatggagg ttaagatcat cgacgcagtc  8161atcggtgaga gaccacctta cttctgcggc ggatttatct tgcaagattc ggttacttcc  8221acagcgtgcc gcgtggcgga tcccctgaaa aggctgttta agttgggtaa accgctccca  8281gccgacgacg agcaagacga agacagaaga cgcgctctgc tagatgaaac aaaggcgtgg  8341tttagagtag gtataacagg cactttagca gtggccgtga cgacccggta tgaggtagac  8401aatattacac ctgtcctact ggcattgaga acttttgccc agagcaaaag agcattccaa  8461gccatcagag gggaaataaa gcatctctac ggtggtccta aatagtcagc atagtacatt  8521tcatctgact aatactacaa caccaccacc tctagagctt gccgccacca tggtgagcaa  8581gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtgaa  8641cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac  8701cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac  8761cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt  8821cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga  8881cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat  8941cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta  9001caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt  9061gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca  9121gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac  9181ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt  9241cgtgaccgcc gccgggatca ctcacggcat ggacgagctg tacaagtaaa gcggccgtga  9301gcatgcaggc cttgggccca atgatccgac cagcaaaact cgatgtactt ccgaggaact  9361gatgtgcata atgcatcagg ctggtacatt agatccccgc ttaccgcggg caatatagca  9421acactaaaaa ctcgatgtac ttccgaggaa gcgcagtgca taatgctgcg cagtgttgcc  9481acataaccac tatattaacc atttatctag cggacgccaa aaactcaatg tatttctgag  9541gaagcgtggt gcataatgcc acgcagcgtc tgcataactt ttattatttc ttttattaat  9601caacaaaatt ttgtttttaa catttcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  9661aaagggaatt cctcgattaa ttaagcggcc gctcgagatg gcacacgtgt tacggtttta  9721ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat  9781tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg  9841ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag  9901tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt  9961ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 10021ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 10081gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 10141gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 10201cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 10261ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 10321tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 10381gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 10441tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 10501ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 10561ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 10621ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 10681accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 10741tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 10801cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 10861taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 10921caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 10981gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 11041gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 11101ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 11161attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 11221gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 11281tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 11341agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 11401gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 11461actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 11521tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 11581attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 11641tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 11701tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 11761aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 11821tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 11881cgcacatttc cccgaaaagt gccacctgac gtc //

What is claimed is:
 1. A virus like particle (VLP) consisting of: a. analphavirus replicon isolated from Sindbis virus or Venezuelan equineencephalitis virus comprising a recombinant polynucleotide comprising asequence encoding Sindbis virus or Venezuelan equine encephalitis virusnonstructural proteins NSP1, NSP2, NSP3, and NSP4; and a retroviralpackaging signal isolated from Rous sarcoma virus, b. a retroviral gagprotein encoded by a polynucleotide comprising a genomic sequence of aRous sarcoma virus, and c. a fusogenic envelope protein, wherein the VLPdoes not contain an alphavirus structural protein gene.
 2. The VLP ofclaim 1, wherein the fusogenic envelope protein is encoded by apolynucleotide comprising a genomic sequence encoding an envelopeprotein selected from the group consisting of haemagglutinin, Roussarcoma virus fusion protein, an E protein of tick borne encephalitisvirus and dengue fever virus, the E1 protein of Semliki Forest virus,baculovirus gp64, VSV-EnvA, and VSV-G protein.
 3. The VLP of claim 1,wherein the VLP is not cytopathic to a eukaryotic cell.
 4. The VLP ofclaim 1, wherein the alphavirus replicon comprises a promoter of a virusRNA-dependent RNA polymerase, wherein said promoter is linked to therecombinant polynucleotide.
 5. The VLP of claim 1, wherein therecombinant polynucleotide encodes an antisense RNA, that knocks downexpression of a gene in the eukaryotic cell.
 6. The VLP of claim 1,wherein the recombinant polynucleotide encodes a shRNA or a miRNA,wherein the shRNA or miRNA knocks down expression of a gene in theeukaryotic cell.
 7. A method of producing a VLP comprising: a.co-transforming a eukaryotic cell with: i. a first vector comprising apolynucleotide sequence encoding the alphavirus replicon, wherein thealphavirus replicon includes the polynucleotide of interest, ii. asecond vector comprising a polynucleotide sequence encoding theretroviral Rous gag protein, and iii. a third vector comprising apolynucleotide sequence encoding the fusogenic envelope protein; b.culturing the co-transformed cell under conditions suitable to causeeach vector to produce its encoded product, thereby producing the VLP;and c. isolating the VLP from the cell; wherein neither the vectors northe cell contain any alphavirus structural protein genes.
 8. A method oftreating or preventing a disease or a disorder in a subject, comprisingadministering to a subject the VLP of claim
 1. 9. The method of claim 8,wherein the vector comprising the polynucleotide sequence encoding thealphavirus replicon is pDest472-VEE or pDest472-VEE-MCS.